Certain closely related species of budding yeast have been shown to contain naturally occurring circular double stranded DNA plasmids. These plasmids, collectively termed 2 μm-family plasmids, include pSR1, pSB3 and pSB4 from Zygosaccharomyces rouxii (formerly classified as Zygosaccharomyces bisporus), plasmids pSB1 and pSB2 from Zygosaccharomyces bailii, plasmid pSM1 from Zygosaccharomyces fermentati, plasmid pKD1 from Kluyveromyces drosphilarum, an un-named plasmid from Pichia membranaefaciens (hereinafter referred to as “pPM1”) and the 2 μm plasmid and variants (such as Scp1, Scp2 and Scp3) from Saccharomyces cerevisiae (Volkert, et al., 1989, Microbiological Reviews, 53, 299; Painting, et al., 1984, J. Applied Bacteriology, 56, 331) and other Saccharomyces species, such as S. carlsbergensis. As a family of plasmids these molecules share a series of common features in that they possess two inverted repeats on opposite sides of the plasmid, have a similar size around 6-kbp (range 4757 to 6615-bp), at least three open reading frames, one of which encodes for a site specific recombinase (such as FLP in 2 μm) and an autonomously replicating sequence (ARS), also known as an origin of replication (ori), located close to the end of one of the inverted repeats. (Futcher, 1988, Yeast, 4, 27; Murray et al., 1988, J. Mol. Biol. 200, 601 and Toh-e et al., 1986, Basic Life Sci. 40, 425). Despite their lack of discernible DNA sequence homology, their shared molecular architecture and the conservation of function of the open reading frames have demonstrated a common link between the family members.
The 2 μm plasmid (FIG. 1) is a 6,318-bp double-stranded DNA plasmid, endogenous in most Saccharomyces cerevisiae strains at 60-100 copies per haploid genome. The 2 μm plasmid comprises a small-unique (US) region and a large unique (UL) region, separated by two 599-bp inverted repeat sequences. Site-specific recombination of the inverted repeat sequences results in inter-conversion between the A-form and B-form of the plasmid in vivo (Volkert & Broach, 1986, Cell, 46, 541). The two forms of 2 μm differ only in the relative orientation of their unique regions.
While DNA sequencing of a cloned 2 μm plasmid (also known as Scp1) from Saccharomyces cerevisiae gave a size of 6,318-bp (Hartley and Donelson, 1980, Nature, 286, 860), other slightly smaller variants of 2 μm, Scp2 and Scp3, are known to exist as a result of small deletions of 125-bp and 220-bp, respectively, in a region known as STB (Cameron et al., 1977, Nucl. Acids Res., 4, 1429: Kikuchi, 1983, Cell, 35, 487 and Livingston & Hahne, 1979, Proc. Natl. Acad. Sci. USA, 76, 3727). In one study about 80% of natural Saccharomyces strains from around the world contained DNA homologous to 2 μm (by Southern blot analysis) (Hollenberg, 1982, Current Topics in Microbiology and Immunobiology, 96, 119). Furthermore, variation (genetic polymorphism) occurs within the natural population of 2 μm plasmids found in S. cerevisiae and S. carlsbergensis, with the NCBI sequence (accession number NC—001398) being one example.
The 2 μm plasmid has a nuclear localisation and displays a high level of mitotic stability (Mead et al, 1986, Molecular & General Genetics, 205, 417). The inherent stability of the 2 μm plasmid results from a plasmid-encoded copy number amplification and partitioning mechanism, which is easily compromised during the development of chimeric vectors (Futcher & Cox, 1984, J. Bacteriol., 157, 283; Bachmair & Ruis, 1984, Monatshefte für Chemie, 115, 1229). A yeast strain, which contains a 2 μm plasmid is known as [cir+], while a yeast strain which does not contain a 2 μm plasmid is known as [cir0].
The US-region contains the REP2 and FLP genes, and the UL-region contains the REP1 and D (also known as RAF) genes, the STB-locus and the origin of replication (Broach & Hicks, 1980, Cell, 21, 501; Sutton & Broach, 1985, Mol. Cell. Biol., 5, 2770). The Flp recombinase binds to FRT-sites (Flp Recognition Target) within the inverted repeats to mediate site-specific recombination, which is essential for natural plasmid amplification and control of plasmid copy number in vivo (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 7270; Jayaramn, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 5875). The copy number of 2 μm-family plasmids can be significantly affected by changes in Flp recombinase activity (Sleep et al, 2001, Yeast, 18, 403; Rose & Broach, 1990, Methods Enzymol., 185, 234). The Rep1 and Rep2 proteins mediate plasmid segregation, although their mode of action is unclear (Sengupta et al, 2001, J. Bacteriol., 183, 2306). They also repress transcription of the FLP gene (Reynolds et al, 1987, Mol. Cell. Biol., 7, 3566).
The FLP and REP2 genes are transcribed from divergent promoters, with apparently no intervening sequence defined between them. The FLP and REP2 transcripts both terminate at the same sequence motifs within the inverted repeat sequences, at 24-bp and 178-bp respectively after their translation termination codons (Sutton & Broach, 1985, Mol. Cell. Biol., 5, 2770).
In the case of FLP, the C-terminal coding sequence also lies within the inverted repeat sequence. Furthermore, the two inverted repeat sequences are highly conserved over 599-bp, a feature considered advantageous to efficient plasmid replication and amplification in vivo, although only the FRT-sites (less than 65-bp) are essential for site-specific recombination in vitro (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 7270; Jayararm, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 5875; Meyer-Leon et al, 1984, Cold Spring Harbor Symposia On Quantitative Biology, 49, 797). The key catalytic residues of Flp are arginine-308 and tyrosine-343 (which is essential) with strand-cutting facilitated by histidine-309 and histidine 345 (Prasad et al, 1987, Proc. Natl. Acad. Sci. U.S.A., 84, 2189; Chen et al, 1992, Cell, 69, 647; Grainge et al, 2001, J. Mol. Biol., 314, 717).
Two functional domains are described in Rep2. Residues 15-58 form a Rep1-binding domain, and residues 59-296 contain a self-association and STB-binding region (Sengupta et al, 2001, J. Bacteriol., 183, 2306).
Chimeric or large deletion mutant derivatives of 2 μm which lack many of the essential functional regions of the 2 μm plasmid but retain functional the cis element ARS and STB, cannot effectively partition between mother and daughter cells at cell division. Such plasmids can do so if these functions are supplied in traces, by for instance the provision of a functional 2 μm plasmid within the host, a so called [cir+] host.
Genes of interest have previously been inserted into the UL-region of the 2 μm plasmid. For example, see plasmid pSAC3U1 in EP 0 286 424. However, there is likely to be a limit to the amount of DNA that can usefully be inserted into the UL-region of the 2 μm plasmid without generating excessive asymmetry between the US and UL-regions. Therefore, the US-region of the 2 μm plasmid is particularly attractive for the insertion of additional DNA sequences, as this would tend to equalise the length of DNA fragments either side of the inverted repeats.
This is especially true for expression vectors, such as that shown in FIG. 2, in which the plasmid is already crowded by the introduction of a yeast selectable marker and adjacent DNA sequences. For example, the plasmid shown in FIG. 2 includes a β-lactamase gene (for ampicillin resistance), a LEU2 selectable marker and an oligonucleotide linker, the latter two of which are inserted into a unique SnaBI-site within the UL-region of the 2 μm-family disintegration vector, pSAC3 (see EP 0 286 424). The E. coli DNA between the XbaI-sites that contains the ampicillin resistance gene is lost from the plasmid shown in FIG. 2 after transformation into yeast. This is described in Chinery & Hinchliffe, 1989, Curr. Genet., 16, 21 and EP 0 286 424, where these types of vectors are designated “disintegration vectors”. In the crowded state shown in FIG. 2, it is not readily apparent where further polynucleotide insertions can be made. A NotI-site within the linker has been used for the insertion of additional DNA fragments, but this contributes to further asymmetry between the UL and US regions (Sleep et al, 1991, Biotechnology (N Y), 9, 183).
We had previously attempted to insert additional DNA into the US-region of the 2 μm plasmid and maintain its high inherent plasmid stability. In the 2 μm-family disintegration plasmid pSAC300, a 1.1-kb DNA fragment containing the URA3 gene was inserted into EagI-site between REP2 and FLP in US-region in such a way that transcription from the URA3 gene was in same direction as REP2 transcription (see EP 0 286 424). When S150-2B [cir0] was transformed to uracil prototrophy by pSAC300, it was shown to be considerably less stable (50% plasmid loss in under 30 generations) than comparable vectors with URA3 inserted into the UL-region of 2 μm (0-10% plasmid loss in under 30 generations) (Chinery & Hinchliffe, 1989, Curr. Genet., 16, 21; EP 0 286 424). Thus, insertion at the EagI site may have interfered with FLP expression and it was concluded that the insertion position could have a profound effect upon the stability of the resultant plasmid, a conclusion confirmed by Bijvoet et al., 1991, Yeast, 7, 347.
It is desirable to insert further polynucleotide sequences into 2 μm-family plasmids. For example, the insertion of polynucleotide sequences that encode host derived proteins, recombinant proteins, or non-coding antisense or RNA interference (RNAi) transcripts may be desirable. Moreover, it is desirable to introduce multiple further polynucleotide sequences into 2 μm-family plasmids, thereby to provide a plasmid which encodes, for example, multiple separately encoded multi-subunit proteins, different members of the same metabolic pathway, additional selective markers or a recombinant protein (single or multi-subunit) and a chaperone to aid the expression of the recombinant protein.
However, the 6,318-bp 2 μm plasmid, and other 2 μm-family plasmids, are crowded with functional genetic elements (Sutton & Broach, 1985, Mol. Cell. Biol., 5, 2770; Broach et al, 1979, Cell, 16, 827), with no obvious positions existing for the insertion of additional DNA sequences without a concomitant loss in plasmid stability. In fact, except for the region between the origin of replication and the D gene locus, the entire 2 μm plasmid genome is transcribed into at least one poly(A)+ species and often more (Sutton & Broach, 1985, Mol. Cell. Biol., 5, 2770). Consequently, most insertions might be expected to have a detrimental impact on plasmid function in vivo.
Indeed, persons skilled in the art have given up on inserting heterologous polynucleotide sequences into 2 μm-family plasmids.
Robinson et al, 1994, Bio/Technology, 12, 381-384 reported that a recombinant additional PDI gene copy in Saccharomyces cerevisiae could be used to increase the recombinant expression of human platelet derived growth factor (PDGF) B homodimer by ten-fold and Schizosacharomyces pombe acid phosphatase by four-fold. Robinson obtained the observed increases in expression of PDGF and S. pombe acid phosphatase using an additional chromosomally integrated PDI gene copy. Robinson reported that attempts to use the multi-copy 2 μm expression vector to increase PDI protein levels had had a detrimental effect on heterologous protein secretion.
Shusta et al, 1998, Nature Biotechnology), 16, 773-777 described the recombinant expression of single-chain antibody fragments (scFv) in Saccharomyces cerevisiae. Shusta reported that in yeast systems, the choice between integration of a transgene into the host chromosome versus the use of episomal expression vectors can greatly affect secretion and, with reference to Parekh & Wittrup, 1997, Biotechnol. Prog., 13, 117-122, that stable integration of the scFv gene into the host chromosome using a δ integration vector was superior to the use of a 2 μm-based expression plasmid. Parekh & Wittrup, op. cit., had previously taught that the expression of bovine pancreatic trypsin inhibitor (BPTI) was increased by an order of magnitude using a δ integration vector rather than a 2 μm-based expression plasmid. The 2 μm-based expression plasmid was said to be counter-productive for the production of heterologous secreted protein.
Bao et al, 2000, Yeast, 16, 329-341, reported that the KlPDI1 gene had been introduced into K. lactis on a multi-copy plasmid, pKan707, and that the presence of the plasmid caused the strain to grow poorly. In the light of the earlier findings in Bao et al, 2000, Bao & Fukuhara, 2001, Gene, 272, 103-110, chose to introduce a single duplication of KlPDI1 on the host chromosome.
Accordingly, the art teaches the skilled person to integrate transgenes into the yeast chromosome, rather than into a multicopy vector. There is, therefore, a need for alternative ways of transforming yeast.